Immune checkpoint pathways in the ageing immune system and their relation to vasculitides
Hid Cadena, Rebeca
DOI:
10.33612/diss.112111572
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Publication date: 2020
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Hid Cadena, R. (2020). Immune checkpoint pathways in the ageing immune system and their relation to vasculitides. University of Groningen. https://doi.org/10.33612/diss.112111572
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R.D Reitsema1*, R. Hid Cadena2*, S.H Nijhof 2, W.H. Abdulahad 1,2, M.G Huitema 1, D.
Paap1,3, E. Brouwer1, A.M.H. Boots 1# , P. Heeringa2#
1Department of Rheumatology and Clinical Immunology, University of Groningen,
University Medical Center Groningen, Groningen, Netherlands.
2Department of Pathology and Medical Biology, University of Groningen,
Universi-ty Medical Center Groningen, Groningen, Netherlands.
3Department of Rehabilitation Medicine, University of Groningen, University
Medi-cal Center Groningen, Groningen, Netherlands.
*: Contributed equally to this study
#: These authors jointly supervised the work
Submitted
Chapter 2
Effect of age and sex on immune checkpoints expression
and kinetics in human T cells
Abstract
Immune checkpoints (ICs) are crucial molecules in maintaining a proper immune balance. As such, ICs are targeted in various cancers and autoimmune diseases. Even though age and sex are known to have effects on the immune system and are well known to impact development of autoimmune diseases, the interplay between age, sex and T cell IC expression is not known.
To study age-and sex-associated effects on IC expression by human T cells, whole blood of 20 healthy young and 20 elderly males and females was stained for CD3, CD4, CD19, CD45RA, CD25, CD28, PD-1, VISTA, ICOS, ICOSL, CD40 and CD40L. In addition, the kinetics of IC expression was studied in vitro by performing time course experiments on anti-CD3 and anti-CD28 stimulated T cells from young and elderly healthy donors (n=10 each) for up to 90 hours.
Our study revealed an age-associated increase of CD40L by human CD4+ and CD8+ T cells and an age-associated decline of ICOS by CD8+ T cells but not CD4+ T cells. Interestingly, CD40 expression by B cells was found decreased in elderly, suggesting modulation of CD40L-CD40 interaction with age. The kinetics of IC ex-pression revealed differences in magnitude between CD4+ and CD8+ T cells but did not seem to be affected by age and sex. Further phenotypic analysis of CD4+ T cell subsets by CD45RA and CD25 expression revealed an age-associated decrease of PD-1+ CD45RA- (memory) CD4+ T cells and increased expression of CD28 in the CD25-expressing CD4+ T cell subsets. This decrease in PD-1+ memory CD4 T cell fre-quencies with age was found to track with the female sex. Cytomegalovirus (CMV) carriage did not confound these results.
Collectively, our results show that both age and sex modulate expression of immune checkpoints by human T cells. These results could have implications for optimising vaccination and IC immunotherapy and move the field towards precision medicine in the management of elderly patient groups.
Introduction
Age and sex are associated with many changes in immune function and with de-velopment of multiple (auto)inflammatory diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) (1–3). In general, ageing is associated with an impaired immune system resulting in a higher incidence of infections in the elderly (4). Major age-as-sociated changes in the immune system include a decrease in the number of lym-phocytes, especially naive CD8+ T cells, and a decrease in the diversity of the T cell receptor (TCR) repertoire (5,6). In addition, humoral immunity wanes with ageing, which is presumably caused by both decreases in B cells and in the production of high-affinity antibodies (7–9). Compared to age-related changes in immune func-tion, the effect of sex on immune function is less well understood. Nevertheless, it is known that there are multiple differences in the immune system between males and females. Some of these aspects are present throughout life, whereas others only emerge after puberty and disappear with the onset of menopause. This sug-gests that both genetic and hormonal factors play a role (10). In general, females show stronger immune responses, including stronger T-cell responses, which may lead to an increased protection against different pathogens (10,11). In addition, fe-male sex hormones such as estrogen enhance B cell responses (12). Consequently, a more active immune system in females during the reproductive years might be prone to develop inflammation and autoimmune related conditions (13).
Immune checkpoints (ICs) are pivotal molecules in the regulation of the im-mune response and thus important when studying age- and sex- associated effects on the immune system. IC molecules are currently targeted to treat cancer and chronic infectious diseases. During chronic infection and cancer, T cells become ex-hausted, a state of poor effector function, which can be reversed by immunothera-py which involves the antagonistic targeting of inhibitory ICs (14). Unfortunately, by activating the immune system to boost the immune response to tumour cells, sev-eral immune-related adverse events (irAEs) affecting multiple organs of the body can occur (15). Among these irAES, development of rheumatic diseases has been reported (16,17), which underlines the importance of ICs in inflammatory diseases and adds to the complexity of IC therapy.
The best studied ICs belong to the so called B7 family, which consists of the co-stimulatory ICs CD28, Inducible T cell costimulatory (ICOS) and co-inhibitory ICs programmed death 1 (PD-1) and V-domain Ig suppressor of T cell activation (VISTA) (18). Except for CD28 expression, limited data is available on effects of age and sex on IC expression. Regarding ageing effects, several studies report on decreased CD28 expression by CD8+ and to a lesser extent CD4+ T cells of elderly people.
Lack of CD28 expression by T cells is considered to be a hallmark of immunosenes-cence. On the other hand, increased PD-1 expression by CD8+ T cells was observed in elderly people as well, which is thought to be associated with a state of early exhaustion (5,19–21). VISTA is a relatively unexplored co-inhibitory IC. Our group recently found that VISTA suppresses T helper 1, T helper 17 and T follicular helper lineage differentiation (22). In addition, given that humoral immunity wanes with ageing, we were also interested in ICs involved in T-B cell interaction and how these are modulated by age and sex. When CD40 on B cells binds to CD40L on activated CD4+ T cells, B cells proliferate, B cell memory is induced and isotype switching and immunoglobulin production occurs (23,24). The pairing of ICOS and ICOSL in T-B cross talk has more indirect effects on B cell functionality as ICOS-ICOSL interaction leads to generation of T follicular helper (Tfh) cells, which are important for the differentiation of B cells into plasma cells (25–28).
The aim of this study was to determine whether age and sex affect IC expres-sion by T cells and if age and sex affect the kinetics of IC expresexpres-sion following in vitro stimulation. To this end, we investigated expression and kinetics of the co-stimula-tory molecules CD28, ICOS and CD40L and the co-inhibico-stimula-tory molecules PD-1 and VISTA on both CD4+ and CD8+ T cells in young and elderly males and females. In ad-dition, we investigated IC expression by memory and regulatory CD4+ T cell subsets and ICOSL and CD40 expression by B cells. Age- and sex- dependent differences in IC expression may underlie the higher propensity of females to develop inflammation and autoimmune conditions. In addition, the knowledge obtained could be import-ant for optimising current vaccination and immunotherapies in the elderly and aid the development of precision medicine.
Materials and methods Study population
To study the effect of age and sex on immune checkpoint expression, 20 healthy young (age <31 years, male/ female ratio 9/11) and 20 healthy elderly donors (age >54 years, male/female ratio 9/11) were enrolled in this study (Table 1). Due to technical constraints, ICOS expression was measured in 13 of the 20 healthy young donors (male/female ratio 7/6). The kinetics of immune checkpoint expression was determined in a subset of donors consisting of 10 healthy young and 10 healthy old donors (median age 26 and 72.5 years), each group consisting of 5 males and 5 females.
The participants health status was confirmed by a clinician through question-naires in young donors and by physical examination, lab tests and questionquestion-naires in elderly donors. Donors need to fulfill the adapted SENIEUR criteria for health status (29). The constitutional review board of the UMCG approved this study (METc2012/375) and all donors gave their written informed consent prior to blood withdrawal.
*= CMV serostatus was inconclusive for 1 of the 20 young donors
Quantification of leukocytes
Absolute leukocyte counts of the majority of donors (14 young and 14 elderly do-nors) were measured in EDTA blood according to the MultiTest TruCount method (BD Biosciences, Durham, NC, USA) according to the manufacturer’s instructions.
When absolute counts of an individual donor were measured on multiple days or when the individual donor’s age differed among the different experiments, the ab-solute counts and ages were averaged (Supplementary Table 1).
CMV status
An in-house Enzyme-Linked Immuno Sorbent Assay (ELISA) test was used to de-termine the CMV status for each donor. To this end, 96-well ELISA plates (Greiner, Kremsmünster, Austria) were coated overnight with lysates of CMV-infected fibro-blasts and control-wells with lysates of non-infected fibrofibro-blasts. Next, serial dilu-tions of serum samples and standard IgG+ sera were incubated for 1 hour. After this, goat anti-human IgG-HRP (Southern Biotech, Birmingham, AL, USA) was added followed by a 1 hour incubation step. TBE substrate (Sigma-Aldrich, St. Louis, MO, USA) was added and samples were incubated for 15 min., after which H2SO4 was used to stop the reaction. The plates were scanned on a Versamax reader (Molecu-lar Devices, Sunnyvale, CA, USA) and data was analysed with SoftMax Pro.
Immune checkpoint expression by whole blood immune cells
To measure IC expression on circulating immune cells, fresh blood collected in EDTA tubes was washed twice with PBS and stained with monoclonal antibodies detecting CD3, CD4, CD45RA, CD25, CD19, CD28, PD-1, VISTA, ICOS, ICOSL, CD40 and CD40L for 15 minutes (Supplementary Table 2A). After surface staining, cells were fixed and red blood cells were lysed with FACS lysing solution (BD Biosciences, 1:10 dilution). Samples were measured on a BD LSR-II flow cytometer. Positivity was determined by isotype controls and IC expression is presented as the percentage of positive cells within CD4+ and CD8+ T cells and B cells. CD4+ T cells were further subclassified into seven fractions by virtue of CD45RA and CD25 expression, based on the classification criteria reported by Miyara et al.(2009) and adapted by van der Geest et al. (2014) (Supplementary figure S1) (30,31). Fraction 1, 2 and 3 identify resting/naive regulatory T cells (Tregs), activated/memory Tregs and non-suppres-sive or cytokine secreting Tregs, respectively. Fraction 4 and 5 comprise memory cells with dim CD25 and memory cells lacking CD25 expression, respectively. Frac-tion 6 and 7 comprise naive cells and age-associated naive CD25dim T cells, respec-tively (Supplementary figure S1).
Kinetics of immune checkpoint expression in vitro
IC kinetics was determined by measuring expression at defined time points after T cell stimulation in vitro. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood collected in heparin tubes by density gradient centrifugation using Lymphoprep (Alere Technologies AS, Oslo, Norway). Next, T cells were iso-lated from PBMCs by negative selection using the MagniSort Human T cell Enrich-ment Kit (Thermo Fisher Scientific, Waltham, MA., USA) or the EasySepTM Human T cell isolation kit (Stemcell Technologies, Vancouver, Canada) (Supplementary Table 1, results were found to be similar for both kits, purity ≥92%) according to man-ufacturer’s instructions. After T cell enrichment, 0.5 x 106 T cells were added to 1 mL of Roswell Park Memorial Institute (RPMI) culture medium with HEPES and L-Glutamine (Lonza, Basel, Switzerland) supplemented with gentamycin (Lonza), in round-bottomed polypropylene tubes. Gibco DynabeadsTM Human T-activator anti-CD3/anti-CD28 (ThermoFisher, Waltham USA) were added to T cells in a ratio of 1:5. Stimulated T-cells were then incubated at 37°C, 5% CO2 and collected after 1,2,3,4, 18, 42, 66 and 90 hours and stained for CD8, CD45RA, CD25, PD-1, VISTA, ICOS and CD40L. In parallel, unstimulated T cells were also assessed for the same ICs including CD28 at each time point (See supplementary Table 2 for the antibod-ies used). Staining was performed as described in the previous paragraph.
Data analysis and statistics
Flow cytometry data was analysed with Kaluza Analysis Software (Beckman Coulter, California, USA) and graphs were created with GraphPad Prism 7 (GraphPad Soft-ware, San Diego, USA). Two-tailed Kruskal- Wallis tests were performed when mul-tiple groups were compared and Mann-Whitney U tests for comparing two groups. Interaction effects of age and CMV status were explored via factorial ANOVA. Age and sex effects on IC kinetics were explored as follows. First, data was plotted in Graph Pad Prism version 7 and visually assessed for a difference in expression of IC expression (Y-axis) between young and elderly males and females over time (X-axis). Consequently, as we visually detected an effect of ageing on PD-1 expression within stimulated CD8+ T cells, these ageing effects were further explored. The peak up-regulation of PD-1 was after 18 hours and gradually declined thereafter but did not reach 0. Therefore, in the subsequent analysis we only took into account measure-ments after 18 hours. We performed a 2-level, multilevel analysis (autoregressive 1st order covariance structure) in SPSS version 23 to analyse if PD-1 expression on CD8+ T cells differed between young and elderly. The highest level was PD-1 ex-pression on CD8+ T cells and the lowest level was the repeated measurements over
time. The dependent variable was PD-1 expression and the independent variables were time of measurement (18, 42, 66 and 90 hours) and age (young or elderly group). Independent factors were entered in the analysis. Interaction effects be-tween age and time, age and stimulation, time and stimulation, time/time2 and stimulation were checked to see whether it improved the model fit. If this was the case, the predictor interaction remained in the equation. Thereafter residuals were checked for normal distribution. After cube root transforming of PD-1 expression, residuals were normally distributed. All results were considered statistically signifi-cant when p<0.05.
Results
Effects of ageing and sex on numbers of circulating immune cells
As ageing has been associated with alterations in peripheral blood immune cell counts, we first determined absolute leukocyte counts in the young and elderly donors by TruCount (Figure 1). We confirmed decreases of total lymphocytes in elderly donors (p=0.039). Furthermore, total T cell (CD3+) numbers tended to be decreased which was mainly due to decreases in CD8+ (p=0.017) but not CD4+ T cells. In addition, B cell numbers were decreased (p=0.004).
Figure 1. Absolute cell counts of total lymphocytes, CD3+, CD4+ and CD8+ T cells, NK cells and B cells in peripheral blood of young and elderly healthy donors. Absolute cell counts
were determined by TruCount, see materials and methods. Horizontal bars reflect median values. Light pink values represent ranges outside the reference range, obtained from the lo-cal diagnostic department. Solid circles represent females and open circles represent males. The Mann-Whitney U test was used for comparisons between young and elderly donors.
Next, whole blood staining of CD4+ T cells employing CD4, CD45RA and CD25 revealed shifts in naive/ memory ratios in the elderly group, as elderly donors had a higher frequency of memory CD4+ T cells (CD45RA-) than young donors (p=0.029) (Supplementary figure S2). Of note, the selected markers (CD45RA and CD25) were chosen to analyse CD4+ T cell differentiation subsets but do not allow an accurate analysis of the CD8 memory and naive subsets.
Furthermore, since CMV serostatus has known effects on immune function and as CMV seropositivity increases with age (32), CMV serostatus was determined for all donors. As expected, more elderly individuals were CMV positive compared to young donors (Table 1). Taken together, these results show ageing-induced
differ-ences in immune composition and CMV serostatus. Importantly, no differdiffer-ences in immune cell numbers were detected between males and females.
Effects of ageing on immune checkpoint expression in circulating CD4+ and CD8+ T cells
To study the effects of ageing on IC expression, frequencies of CD28+, PD-1+, VIS-TA+, ICOS+ and CD40L+ T cells were first determined within total circulating CD4+ and CD8+ T cells of young and elderly donors. Ageing did not have a strong effect on frequencies of CD28+, PD-1+ and VISTA+ cells within both CD4+ and CD8+ T cell populations (Figure 2). However, ageing affected CD40L expression, as a statistically significant increase in the frequency of CD40L+ cells within both CD4+ and CD8+ T cells was observed in elderly donors, respectively (p<0.0001 and p<0.001). In addi-tion, frequencies of ICOS+ cells within CD8+ T cells were decreased in elderly do-nors (p<0.001), whereas ICOS+ CD4+ T cell frequencies did not seem to be affected by age.
Figure 2. Immune checkpoint expression frequencies within CD4+ and CD8+ T cells of young and elderly donors. Ageing effects on immune checkpoint expression were
deter-mined by flow cytometric staining of whole blood. Graphs represent percentages of CD28+, PD-1+, VISTA+, ICOS+ and CD40L+ cells within total CD4+ (A) and CD8+ T cells (B). Open and closed circles represent males and females, respectively. Horizontal bars reflect median per-centages. The Mann-Whitney U test was used for comparison between young and elderly donors. P values are indicated in the graphs.
Effects of ageing on ICOSL and CD40 expression by circulating B cells
Both CD40/CD40L and ICOS/ICOSL interactions are important in T and B cell cross-talk. As we observed a proportional increase of CD40L expression by CD4+ T cells in elderly donors but no effects of ageing on ICOS expression by CD4+ T cells, we wondered how ageing affects CD40 and ICOSL expression by B cells. Interestingly, whereas frequencies of ICOSL expressing B cells were not different between young and elderly donors, a proportional decrease of CD40+ B cells was found in elderly donors (n=16) (Figure 3). Thus, age had an opposite effect on CD40L expression by T cells and CD40 expression by B cells.
Figure 3. CD40 and ICOSL expression by B cells of young and elderly donors. Ageing effects
on CD40 and ICOSL expression were determined by flow cytometric staining of whole blood. Graphs represent percentages of CD40+ cells (A) and ICOSL+ cells (B) within total B cells (A). Open and closed circles represent males and females, respectively. Horizontal bars reflect median percentages. The Mann-Whitney U test was used for comparison between young and elderly donors. P values are indicated in the graphs.
Effects of ageing on immune checkpoint kinetics in circulating CD4+ and CD8+ T cells
To investigate whether the capacity to express ICOS, CD40L, PD-1 and VISTA by CD4+ and CD8+ T cells upon stimulation is affected by ageing, we stimulated en-riched CD4+ and CD8+ T cell populations with anti-CD3 and anti-CD28 stimulation beads and assessed proportions of IC positive cells at 1, 2, 3, 4, 18, 42, 66 and 90 hours thereafter. Figure 4A illustrates the kinetics of checkpoint expression by CD4+ T cells of young and elderly donors. First, CD40L was most promptly upregulated and peaked at 18 hours after stimulation with more than 60% of CD40L+ T cells,
af-ter which frequencies gradually declined over time. The kinetics of PD-1+ and ICOS+ cells showed a somewhat slower proportional increase, and expression reached a plateau at around 40% of CD4+PD1+ cells. The frequency of VISTA+ cells did not follow a clear pattern of upregulation after stimulation and remained low (<10%) compared to the other ICs. No effects of age on IC expression kinetics by stimu-lated CD4+ T cells was detected. In addition, we did not detect differences between males and females on the kinetics of IC expression (data not shown). This would suggest that the capacity of T cells to upregulate immune checkpoints after anti-genic stimulation is stable over age and comparable between males and females.
Figure 4: Kinetics of immune checkpoint expression. T cells were stimulated and immune
checkpoint expression was measured at several time points after T cell stimulation. Graphs illustrate median percentages of PD-1, VISTA, ICOS and CD40L expression by total CD4+ T cells (A) and CD8+ T cells (B) at indicated time points (n=10 young and 10 elderly donors). Black and grey solid lines represent the median expression percentages of young and elderly donors, respectively. Dotted black and grey lines represent unstimulated cells. Black circles represent young donors and triangles elderly donors. Error bars indicate interquartile range.
The pattern of IC upregulation by CD8+ T cells was less pronounced than seen with the CD4+ subset (Figure 4B). Also, whereas the frequency of PD-1+ cells within CD4+ T cells stabilized after 42 hours of stimulation, their frequencies within CD8+ T cells decreased somewhat after 42 hours. Of note, CD8+ T cells did not seem to upregulate VISTA at all. In contrast, whereas age did not affect the kinetics of stim-ulation-induced PD-1 expression by CD4+ T cells, it appeared that PD1+ frequencies within CD8+ T cells were more readily induced in the elderly. This difference, how-ever, did not reach statistical significance as there was no main effect of ageing in the multi-level analysis (Table 2).
a: estimates of fixed effects b: cube transformed values
Effects of Ageing on IC expression by CD4+ T cell differentiation subsets
Our data confirmed increased memory/naive T cell ratios in elderly donors. In ad-dition, in line with previous studies, our further subtyping of naive and memory fractions, using the Miyara classification based on CD45RA and CD25 expression, revealed a shift from fraction 6 (naive T cells) to the ageing-associated fraction 7 (naive, CD25dim T cells) in elderly donors (30,33) (Supplementary figure S3). Given these compositional changes, we next analysed the expression of ICs on naive and memory effector CD4+ T cell fractions (fractions 4-7, Supplementary figure S1). In addition, we assessed IC expression on naive and memory Treg subsets (fractions 1-3) to gain additional insight in IC expression by effector versus regulatory CD4+ T cells.
Interestingly, the higher frequencies of CD40L+ cells within CD4+ T cells of elderly individuals (Figure 2) was characteristic of all CD4+ fractions although fre-quencies were relatively low (ranging between 0.4-4%, Supplementary figure S6). No effects of age were found on ICOS expression within total CD4+ T cells, as opposed to CD8+ T cells. After further subsetting of CD4+ T cells, however, frequen-cies of ICOS+ cells within naive CD4+ T cells were found to be decreased in elderly donors, although the differences were small (Supplementary figure S7).
It is well known that CD28- cells accumulate with ageing, especially in CD8+ T cells and to a lesser extent in CD4+ T cells (21). Whereas in our study the frequen-cies of CD28+ cells within total CD4+ and CD8+ T cells were not affected by age in these selected healthy elderly donors, some effects of ageing were found after further subsetting of CD4+ T cells (Supplementary figure S5). We observed an unex-pected proportional increase of CD28+ cells in the CD25-expressing T cell fractions of elderly donors. More specifically, in fractions 4 (p=0.001) and 7 (trend p=0.068) and in Treg fractions 1, (p<0.0001), 2 (p<0.0001) and 3 (p=0.001). Our data thus link increased frequencies of CD4+CD28+ in elderly to all CD25-expressing CD4+ subsets including Treg, suggesting a higher state of activation.
Furthermore, whereas PD-1 frequencies did not seem to be affected by age within total CD4+ T cells, subsetting of CD4+ T cells revealed strong effects of age on PD-1+ frequencies among naive and memory CD4+ T cells including the Tregs (Figure 5). More specifically, in the elderly donors, the frequencies of PD-1+ cells were decreased among total memory CD4+ T cells (p=0.04), fraction 4 (p=0.003) and Treg fractions 3 (p=0.011) and 2 (p=0.038). Also, PD-1 expression within frac-tion 7 was decreased upon ageing (p=0.005), whereas PD-1 expression in fracfrac-tion 1, the resting/naive Tregs, was increased (p=0.003). The latter may have obscured the effects of age on PD-1 expression of total CD4+ T cells. Of note, VISTA expres-sion frequencies among different CD4+ T cell fractions were not affected by ageing (Supplementary figure S8).
Collectively, a more detailed analysis of CD4+ T cell differentiation subsets revealed ageing associated modulation of CD28, CD40L and PD-1 expression fre-quencies among both effector and regulatory subsets.
Figure 5: PD-1 expression frequencies among naive and memory fractions of CD4+ cells of young and elderly donors. Ageing effects on PD-1 expression were determined by flow
cytometric staining of whole blood. Graphs represent percentages of PD-1+ cells within to-tal naive and different naive fractions and toto-tal memory and different memory fractions of CD4+ T cells. Open and closed circles respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from reg-ulatory fractions. The Mann-Whitney U test was used for comparison between young and elderly donors. P values are indicated in the graphs.
Effect of CMV serostatus on immune checkpoint expression by circulating CD4+ and CD8+ T cells
Ageing is associated with an increase in CMV seropositivity. Also, it is well known that CMV carriage modulates the immune system. Indeed, oligoclonal expansions of especially late stage memory CD8+ and presumably also CD4+ memory cells is typical of CMV infection (34). Moreover, higher frequencies of both CD8+ and CD4+ T cells lacking the IC CD28 have been reported in CMV carriers (35,36). Consequent-ly, carriage of CMV may modulate expression of other IC molecules and should be excluded as a confounder in our study. Therefore, we assessed whether there are interaction effects of age and CMV in modulation of ICs by total CD4+ and CD8+ T cells and by CD4+ T cell subsets.
Frequencies of CD40L were found increased within total CD4+ and CD8+ T cells of elderly donors. A statistically significant interaction effect between CMV (n= 19 young, n=20 elderly) and age was found for CD40L expression in CD4+ T cells (F(1, 35) = 5.2, p <0.05) and a trend in CD8+ T cells (F(1, 35) = 4.1, p=0.052). Comparison
of CD40L expression by CD4+ and CD8+ T cells between young and elderly CMV+ and CMV– carriers, however, showed that CMV carriage seems to dampen the age-associated proportional increases of CD40L+ T cells (Supplementary figure S4). Ageing effects on CD40 expression by B cells were not confounded by CMV status. CD28 was not differently expressed by total CD4+ and CD8+ T cells between young and elderly donors. After CD4+ subsetting, however, a proportional increase in CD28 expression was noted in the CD25+ fractions of CD4+ T cells of elderly do-nors. Interestingly, an interaction effect of CMV and age was found regarding CD28 expression within fraction 3 (resting/ non suppressive Tregs, F(1, 35) = 4.6, p<0.05) and fraction 4 (memory CD25dim cells, F(1, 35) = 4.3, p < 0.05). Contrary to expecta-tions, comparison of CD28 expression in these fractions between elderly and young CMV+ and CMV- carriers shows that CD28 expression is increased upon ageing in CMV+ donors (and thus CD28- cells decreased) but not in CMV- donors ( Supple-mentary figure S4).
ICOS was especially decreased by CD8+ T cells in elderly but an interaction be-tween CMV and age was not found. Furthermore, PD-1 expression was decreased within several subsets of CD4+ T cells in elderly donors. Also, in these subsets no interaction effects between CMV and age were found.
Taken together, age associated effects on ICOS, PD-1 and CD40L expression by CD4+ T cells are not likely confounded by CMV carriage. In contrast, there was an interaction effect of age and CMV serostatus for CD28 expression in defined CD4+ subsets. Here, CMV carriage led to proportional increases of CD4+CD28+ in elderly donors, a finding to be further investigated.
Effects of sex on immune checkpoint expression by circulating CD4+ and CD8+ T cells
Sex effects on IC expression were determined by comparing IC expression by CD4+ and CD8+ T cells between males and females within each age group. Whereas sex did not affect CD28, VISTA, ICOS and CD40L expression frequencies within total CD4+ and CD8+ T cells and PD-1 within CD8+ T cells (data not shown), sex did have a substantial effect on the expression of PD-1 by (fractions of) CD4+ T cells. Inter-estingly, the frequency of PD-1+ cells differed especially between elderly females and males (Figure 6A). Elderly females had lower frequencies of PD-1+ cells than males within total CD4+ T cells (p=0.038), total memory CD4+ T cells (p=0.046) and the memory fraction 5 (p=0.038), fraction 4 (p=0.02) and the non- suppressive Treg memory fraction 3 (p=0.031). Fraction 2, comprising activated Tregs, was the only memory fraction in which PD-1 expression did not differ between elderly males and females.
As our subsetting analysis revealed decreases of PD-1 frequencies in overlap-ping memory CD4+ CD25dim cells (fraction 4) and the non-suppressive Tregs (frac-tion 3) in elderly donors compared to young donors, we aimed to assess whether the age-associated decrease in these fractions is solely dependent on female sex. To this end, we assessed whether PD-1 expression was different between young fe-males and elderly fefe-males (Figure 6B and 6C). Indeed, the age-associated decline in PD-1 frequencies in fractions 3 and 4 was found to associate with female sex since elderly females, but not elderly males, had a lower frequency of PD-1+ cells than young females (fraction 3, p=0.004 and fraction 4, p=0.003, Figure 6B,C).
Taken together, our data reveal an age- associated decline of CD4+PD-1+ frequen-cies within defined effector memory subsets of elderly females only.
Figure 6: Effect of sex on PD-1 expression. Sex effects on PD-1 expression were determined
by flow cytometric staining of whole blood. Graphs show the frequencies of PD-1+ cells in elderly males and females within different CD4+ fractions (A), and in all groups in fraction 3 (B) and fraction 4 (C). Open and closed circles in figure 5A respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regulatory fractions. For the comparison of 4 groups, a Kruskal-Wallis test was performed and found to be statistically significant in fraction 3 and 4 (p=0.019 and p=0.005). The Mann-Whitney U test was used for comparison between two groups. P values are indicated in the graphs.
Discussion
In this study we show that both age and sex modulate expression of immune check-points by human T cells. More specifically, our study revealed an age-associated increase of CD40L by human CD4+ T cells and an age-associated decline of PD-1 expression by CD4+ memory T cells of elderly females. The latter finding may aid the optimisation of PD-1 targeted immunotherapy and help the implementation of precision medicine in management of this vulnerable patient group.
Given the limited knowledge on effects of age and sex on IC expression, this study aimed to investigate checkpoint expression and induction kinetics in young and elderly healthy males and females. Interestingly, the effects of age on T cell PD-1 expression were not conspicuous and were only revealed after subsetting of CD4+ T cells using CD45RA and CD25, adding to the value of this classification meth-od (30). By measuring IC expression within defined subsets of conventional and reg-ulatory CD4+ T cells, we found that PD-1 expression by especially effector memory CD4+ T cell subsets is affected by both age and sex. More specifically, PD-1 expres-sion within defined fractions of memory CD4+ T cells is decreased upon ageing and is lower in elderly females than in elderly males. Stratifying results according to sex revealed also that the observed ageing effect on the non-suppressive and cytokine secreting Tregs (fraction 3) and the effector memory CD25dim cells (fraction 4) can be attributed solely to the decrease of PD-1 in elderly females. Previously, we re-ported on higher frequencies of PD-1 expressing CD4+ T cells in young but not old patients with metastatic melanoma, data consistent with the current finding albeit that effects of sex were not documented (37).
PD-1 has been identified as a crucial IC in the regulation of the immune system and more specifically in preventing auto-immune diseases (38). The rationale for this association arose from observations in PD-1 knockout mice, since these mice were prone to develop auto-immune diseases (39,40). Furthermore, blocking PD-1 in an experimental autoimmune encephalomyelitis (EAE) mouse model resulted in aggravated disease progression (41).
Since elderly females in this study likely reached the post-menopausal status, the observed decrease in PD-1 frequency in elderly females compared to young females, may be explained by a decrease in female sex hormones such as estro-gens. Interestingly, previous studies in mice have suggested an effect of estrogen on PD-1 expression by CD4+ Tregs. Firstly, estrogen (17-beta-estradiol) treatment increased intracellular PD-1 expression in CD4+ Forkhead box P3 (FOXP3)+ (Treg) cells in mice whereas oestrogen receptor knockout (ERKO) mice had reduced PD-1 expression and reduced Treg suppression (42). Secondly, the PD-1 induction in the Treg compartment by estrogen was correlated with better suppression and hence
EAE protection whereas Treg deficient mice appeared to be unprotected against spontaneous EAE. Lastly, estrogen reduced IL-17 levels in immunized mice, whereas estrogen did not reduce IL-17 levels in PD-1 deficient mice. These results suggest suppression of IL-17 by estrogen-induced PD-1+ Tregs (43).
Thus, the mouse studies show that PD-1 expression by Treg can be modulated by estrogens with clear consequences for experimental autoimmunity. It remains to be elucidated if female sex hormones modulate T cell PD-1 expression in humans and if this is limited to the Treg compartment. Furthermore, further studies are war-ranted to investigate whether female sex hormones in relation to PD-1 expression are associated with the higher incidence of auto-immune diseases in females. Addi-tionally, other (hormonal) factors are likely involved in the regulation of T cell PD-1 expression as well, as we did not observe lower PD-1 frequencies in elderly males, suggesting that effects of androgens and DHEAS, the main androgen in women, should be studied as well. Nevertheless, some auto-inflammatory diseases such as Giant Cell Arteritis (GCA), a large vessel vasculitis typically affecting elderly females, and late-onset Rheumatoid Arthritis (RA) could be related to a post-menopausal decrease in PD-1 expression. Clearly, this would require further dedicated studies. Importantly, age- and sex- associated differences in PD-1 expression could have implications for PD-1 checkpoint blockade therapies in cancer. These therapies typically reverse the state of exhaustion of tumour specific effector T cells. Several papers have reviewed the efficacy of PD-1 blocking therapies in clinical trials having included older adults and found contradictory and inconsistent results. For some cancers such as head and neck, non-small cell lung cancer (NSCLC) and metastatic renal cell carcinoma (mRCC), the efficacy of IC therapies in elderly people seemed to be decreased (44), but often no age-associated differences were found (45,46). In addition, some studies noted increased efficacies with ageing (47). Overall, it should be appreciated that elderly patients (>65 years) have been under-represented in many clinical trials, and that those who were included may not be representative of the elderly population in general (44,48). In addition, a meta-analysis addressing sex-dependent effects of IC therapy revealed a lower efficacy in women than in men. Here, differences between young and elderly patients were not addressed (49). Together, these findings indicate that more studies are needed to determine the efficacy and toxicity (iRAEs) of IC therapy in young and elderly males and fe-males. Assessment of T cell PD-1 expression before start of treatment and how this affects efficacy and safety of PD-1 blockade therapy remains to be studied.
Frequencies of CD40L+ T cells were impacted by ageing as well. Although fre-quencies of CD40L+ cells were low and differences in frefre-quencies between young and elderly people appeared to be subtle, the increase in the elderly group was con-sistent and seen in every subset of CD4+ T cells. CD40L is important in T cell/ B cell
interactions, and an increased expression could lead to an increased B cell response (23,24). However, this is contradictory with the notion that the humoral response wanes with ageing. Indeed, the observed proportional increase in CD40L+ cells seems to be a compensatory mechanism as CD40+B cells were decreased upon age-ing. CD40L/CD40 interactions have been found to be important in autoimmune dis-eases such as SLE. In SLE, CD40L expression is increased on circulating B and T cells but data on CD40 expression levels have not been reported (50). Elevated CD40L levels have been suggested to cause an amplification of the immune response and an increase in autoantibodies (50–52). Whether the observed increase of CD40L by circulating CD4+ T cells in elderly donors is solely a compensatory mechanism or has consequences for immune function and humoral responses remains to be further investigated.
Previous studies have shown a decrease in CD28 expression by CD8+ T cells and to a lesser extent CD4+ T cells, which has been attributed to both ageing and CMV carriage (5,35,53). Unexpectedly, CD28 positivity was not decreased but rath-er increased upon ageing in this study, especially in CD4+ Tregs. To investigate why we did not find decreases in CD28+ cells, we analysed whether there was an inter-action effect of CMV and age. Indeed, an interinter-action effect of age and CMV for CD28 expression was found within non-suppressive Tregs and CD25dim memory CD4+ T cells. That is, the observed proportional increase of CD28 upon ageing was solely visible within CMV carriers. The reason for this is currently unknown, since other well-known phenomena associated with ageing such as a decrease in lymphocytes, CD8+ T cells, B cells and an increase in memory CD4+ T cells were confirmed in this study (6,54). The increased CD28+ frequencies in elderly donors were especially found within CD4+ Tregs suggesting increased suppressive capacity of Tregs in el-derly. However, even though Treg numbers increase with age, studies did not report increased Treg function in elderly donors (55,56). Thus, the implication of the ob-served increased CD28+ frequencies in activated and resting Tregs of elderly donors remains to be elucidated.
The determination of IC kinetics clearly shows that IC kinetics appeared to be different between CD4+ and CD8+ T cells. In line with our observations, an in vitro study performed by Sabins et al. showed higher PD-1 expression by CD4+ T cells than by CD8+ T cells (57). Although differences are seen in ICs expression by circu-lating T cells, the capacity of T cells to upregulate IC expression upon stimulation in
vitro appears to be unaffected by age and sex.
This study has some limitations. First, we report results of a relatively small number of healthy donors. Results in this study should therefore be interpreted with caution and be seen as a first step towards more information on age and sex effects on IC expression and kinetics. Also, our study was not designed and
pow-ered to definitely conclude on effects of CMV carriage. Our retrospective analysis, however, suggests that the effects of age and sex on CD40L and PD-1 expression frequencies are not confounded by CMV carriage. Secondly, while assessing the kinetics of IC expression, we did not determine other functional markers such as proliferative capacity and cytokine production in our assays, which could have re-vealed more clues as to how age and sex affect T cell function. Furthermore, CD25 and CD45RA expression did not allow for more detailed subsetting of CD8+ T cell populations, as it is designed for closer inspection of CD4+ T cell subsets. Subsetting of CD8+ T cells could be important, since some ageing effects were not found on total CD4+ T cells but became apparent only after further subsetting. Lastly, our methods did not allow for accurate analysis of per cell IC expression (MFI), which could give additional information when analysing the effects of combined positive and negative IC expression.
Despite these limitations, we provide evidence that both age and sex modu-late IC expression by human T cells. We hope that our findings will prompt further research as more knowledge may aid the optimisation of vaccination and targeted immunotherapy and move the field towards precision medicine in the management of elderly patient groups.
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Supplementary Table S1: Overview of the Study population
X: Absolute counts determined for indicated experiment.
O : Absolute counts determined on days when checkpoint expression and kinetics were both measured.
Naive Fractions
Memory Fractions
Supplementary figure S1. Flow cytometric strategy for determining memory and naive CD4+ T cell subsets. By staining for CD45RA and CD25 expression, as reported by Miyara
et al.(2009) (30) and adapted by van der Geest et al.(2014) (33), seven fractions were dis-tinguished. Memory fractions CD45RA-: Memory CD25- (fraction 5), Memory CD25dim (fraction 4), Memory CD25int (fraction 3) and Memory CD25high Treg (fraction 2). Naive fractions CD45RA+: Naive CD25- (fraction 6), CD25dim (fraction 7) and Naive CD25int Treg (fraction 1).
Supplementary figure S2. Memory / naive ratios within CD4+ T cells in young and elderly donors.
The frequencies of memory and naive cells were determined by flow cytometric staining of whole blood. Horizontal bars reflect median percentages. The Mann-Whitney U test was used to compare ratios of memory cells and naive cells between young and elderly donors. P value is indicated in the graph.
Supplementary figure S3. Proportions of memory and naive fractions within CD4+ T cells in young and elderly donors. The frequencies of memory and naive fractions were
deter-mined by flow cytometric staining of whole blood with CD25 and CD45RA. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regula-tory fractions. The Mann-Whitney U test was used to compare frequencies between young and elderly donors. P value is indicated in the graph.
Supplementary figure S4: Effects of CMV serostatus on CD40L and CD28 expression. CMV
effects on immune checkpoint expression were determined by flow cytometric staining of whole blood. Graphs represent percentages of CD40L+ cells within total CD4+ and CD8+ T cells and CD28+ cells within fraction 3 (non-suppressive Tregs) and fraction 4 (CD25dim memory cells). Open and closed circles represent males and females, respectively. Hori-zontal bars reflect median percentages. Kruskal- Wallis test was used for testing between groups after which the Mann-Whitney U test was used for comparison between young and elderly CMV+ and CMV- donors. P values are indicated in the graphs.
Supplementary figure S5. Percentages of CD28+ cells within fractions of CD4+ T cells in young and elderly donors. Ageing effects on CD28 expression were determined by flow
cytometric staining of whole blood. Open and closed circles respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regulatory fractions. The Mann-Whitney U test was used to compare frequencies of CD28+ cells between young and elderly donors. P values are indicated in the graphs.
Supplementary figure S6. Percentages of CD40L+ cells within fractions of CD4+ T cells in young and elderly donors. Ageing effects on CD40L expression were determined by flow
cytometric staining of whole blood. Open and closed circles respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regulatory fractions. The Mann-Whitney U test was used to compare frequencies of CD40L+ cells between young and elderly donors. P values are indicated in the graphs.
Supplementary figure S7. Percentages of ICOS+ cells within fractions of CD4+ T cells in young and elderly donors. Ageing effects on ICOS expression were determined by flow
cytometric staining of whole blood. Open and closed circles respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regulatory fractions. The Mann-Whitney U test was used to compare frequencies of ICOS+ cells between young and elderly donors. P values are indicated in the graphs.
Supplementary figure S8. Percentages of VISTA+ cells within fractions of CD4+ T cells in young and elderly donors. Ageing effects on VISTA expression were determined by flow
cy-tometry. Open and closed circles respectively represent males and females. Horizontal bars reflect median percentages. Dashed line divides memory and naive fractions from regula-tory fractions. The Mann-Whitney U test was used to compare frequencies of VISTA+ cells between young and elderly donors. P values are indicated in the graphs.
